Structure And Functional Studies Of A Novel Chlamydophila Psittaci Cysteine Desulfurase Protein, CPSIT₀959

[Objective]This study was to predict the simulative structure of Chlamydophila psittaci cysteine desulfurase protein CPSIT₀959, and investigate its related functions. [Methods]The structure of CPSIT₀959 protein was analysized and simulated by the homology modeling method of SWISS-MODEL. DNA was extracted from Cps 6BC genome as the temple and specific pairs of Primers were designed by PRIMER5.0. Then CPSIT₀959 gene sequence was amplified by polymerase chain reaction（PCR） and subcloned into vector p ET-30 a in order to construct recombinant plasmid p ET-30a-CPSIT₀959, then transformed the recombinant plasmid into E. coli BL21. The expression of recombinant protein was induced with isopropyl-β-D-thiogalactopyranoside（IPTG） and identifed in Western blotting. The recombinant protein was purified by affinity chromatography using Nickel nitrilotriacetic acid（Ni-NTA） beads.The concentration of recombinant protein was detected by BCA assay. The recombinant protein was used to immunize BALB/c female mice to produce polyclonal antibodies. The titer of polyclonal antibodies was detected by indirect ELISA. The polyclonal antibodies were subsequently used to investigate and analysis the subcellular localization of CPSIT₀959 protein by indirect immunofluorescence assay（IFA）. According to the desulfurization reaction mechanism of cysteine desulfurase, the detection of generated sulfide was used to confirm its desulfurization enzyme activity. [Results] The structure of CPSIT₀959 protein was analysized and simulated. The recombinant expression plasmid p ET-30a-CPSIT₀959 was constructed successfully and the sequencing results were in complete accord with the sequences on Genbank. The recombinant protein was expressed and purified. Specific humoral response was induced by the purified recombinant protein CPSIT₀959 in BALB/c female mice, and the specific antibody titer of CPSIT₀959 reached to 1:512000 which was detected by indirect ELISA. According to the results of IFA, it showed that the distribution pattern of the CPSIT₀959 protein was similar to that of the major outer membrane protein（MOMP）, but not to that of inclusion membrane protein A（Inc A）. According to the desulfurization reaction mechanism of cysteine desulfurase, the CPSIT₀959 protein was confirmed its desulfurization enzyme activity, which can catalyze the conversion of L-cysteine to sulfide, and may be involved in iron–sulfur cluster biogenesis in Chlamydia.[Conclusions]1.CPSIT₀959 protein was successfully expressed and purified in prokaryotic expression system, and it may locate in the Cps inclusion. 2.CPSIT₀959 protein has the desulfurization enzyme activity, which can catalyze the conversion of L-cysteine to sulfide, and may be involved in iron–sulfur cluster biogenesis in Chlamydia.